JPH0477424A - Preventive for periodontal disease - Google Patents

Abstract

PURPOSE:To provide the subject drug containing catechins such as gallic acid, (+)-catechin, (+)-gallocatechin and (-)-epicatechin extracted from tea leaves and having a specific growth inhibitory effect on Bacteroides gingivalis as the pathogenic microorganism of periodontal diseases. CONSTITUTION:A preventive for periodontal diseases, containing catechins such as gallic acid, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, (-)- epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, dimer thereof and trimer thereof extracted from tea as the active components. The above-mentioned catechins can specifically inhibit growth of Bacteroides gingivalis as the pathogenic microorganism of periodontal diseases and have a inhibitory effect on a collagenase produced from the bacteroid. Since the drug can be obtained from tea free from side effects there is no problem of safety. The drug is mixed in a gum, a candy, a tablet candy, a gargle, a tooth paste, etc., to obtain the objective preventive effect.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-periodontal agent effective for the prevention and treatment of periodontal disease. Bacteroides
The present invention relates to an anti-periodontal disease agent which specifically inhibits the growth of C. gingivalis and further inhibits collagenase produced by this bacterium, and which contains a specific component contained in a natural plant as an active ingredient.

[Prior Art] Bacteroides gingivalis has been reported to be frequently isolated from the bottom of periodontal pockets of patients with periodontal disease, and is considered to be a likely pathogen of adult periodontitis, which is the most common cause of periodontal disease. ing. Therefore, in order to improve the symptoms of periodontitis, it is important to inhibit the growth of this bacterium in the lesion, and for this purpose, it is effective to apply antibacterial substances that are effective against this bacterium. it is conceivable that.

Conventionally, antibiotics such as tetracycline and minocycline have been used for this purpose, but although these have strong effects, their use is limited due to the appearance of resistant bacteria and side effects.

The present inventors searched for substances that showed efficacy in a growth inhibition test of Bacteroides gingivalis, which is considered to be a potential pathogen of periodontal disease, using natural products that have been used for a long time and are free from side effects. As a result, it was found that a specific active ingredient that can be extracted from a specific +let exhibits a remarkable specific effect.

Furthermore, this active ingredient also has collagenase inhibitory activity produced by Bacteroides gingivalis, and has been found to be a very effective anti-dental disease agent.

[Problems to be Solved by the Invention] The present invention specifically inhibits the growth of Bacteroides gingivalis, which is contained in a natural product that has been used for a long time without concerns about side effects and is known as a periodontal disease pathogen. Furthermore, it has the effect of inhibiting collagenase produced by this bacterium,
The purpose of the present invention is to provide an anti-periodontal disease agent effective for preventing and treating periodontal disease.

[Means for Solving the Problems] According to the present invention, the periodontal disease pathogen Bacteroides gingivalis
), and further has an inhibitory effect on collagenase produced by Bacteroides gingivalis, which contains gallic acid, (deca)-catechin,
(+)-gallocatechin, (-)-epicatechin, (-)
-epigallocatechin, (=)-epicatechin gallate,
(-)-epigallocatechin kalate and their dimers, trimers, etc.
Provided is an anti-dental disease agent characterized by containing catechins extracted from S) as an active ingredient.

The product of the present invention is an anti-dental disease agent containing tea catechins as an active ingredient. Tea catechins include gallic acid, (10)-chikikin, (±)-gallocatechin, (-)-epicatechin, (
-)-epigallocatechin, (-)-epigallocatechin, (-)-epigallocatechin kalate, dimers, trimers, etc. thereof are intended.

The formula for this kind of tea catechins is as shown below, for example.

gallic acid OH R, HR2H:・ (-)-epicatechin, R, OHR2H: (-)-epigallocatechin, R,H: (+)-catechin OH: (+)-gallocatechin (-)-epicatechin gallate, (-)-Epigallocatechin gallate.

The catechins that can be used in the present invention can be extracted from tea confectionery, for example, and the extraction method is not particularly limited, but it is preferable that the catechins be extracted in a high concentration.

Specifically, extraction from tea confectionery can be performed as follows, for example.

Using 100g of crushed tea confectionery, 80% ethanol solution 2
000ml for 2 hours and dry the extract. The dried extract is diluted with water to a solid content of 10 to 30% and adsorbed on Toyopearl HW-40 gel beads for 1 to 2 hours. At this time, 1 for the volume IJ of gel beads.
It is suitable if the amount of extract is 00 to 300 g. The gel beads adsorbed with the extract are washed 2 to 3 times using 2 to 5 times the amount of water, and the gel beads are further washed 2 to 3 times with 2 to 5 times the amount of 15% ethanol aqueous solution. The product of the present invention can be obtained by liberating the catechins adsorbed on the gel beads using an 80% ethanol aqueous solution and freeze-drying the obtained tea catechins.

The tea catechins of the present invention are specific to Bacteroides gingivalis among oral bacteria. Its antibacterial effect is due to its antibacterial effect, such as Streptococcus, which is a caries-causing bacterium.
mutans, but strong against the periodontal disease pathogen Bacteroides gingivalis.

Tea, which is the raw material for the product of the present invention, is a common drink, and there are no problems with its safety.

Therefore, the product of the present invention can be used for various purposes, and there is no need to worry about side effects. For example, it can be added to chewing gum, candy, mouthwash, toothpaste, etc. for daily use. When the product of the present invention is added to a product, it is preferable to add it in an amount of 0.001 to 10% by weight. .

[Effects] The present invention conducted a growth inhibition test on Bacteroides gingivalis using natural products that have been used for a long time without worrying about side effects, and found a specific effect on tea catechins. It was discovered that tea catechins also have collagenase inhibitory activity produced by Bacteroides gingivalis, and are highly effective anti-periodontal agents. Such effects of tea catechins have not been known in the past, and have been revealed for the first time through the present invention.

Periodontal disease is an inflammatory disease of bacterial origin, and its causes include an increase in periodontal pathogenic plaque bacteria, bacterial tissue invasion, and host response to infection. Bacteroides gingivalis is considered to be the most likely pathogen of adult periodontitis, which is the most common type of periodontal disease, and has been reported to be frequently isolated from the lower periodontal pockets of patients with periodontal disease. There is. This bacterium is a non-motile, Durham-negative bacillus that forms black colonies on blood plates, and much is known about its virulence factors.

Bacteroides, including this bacterial species, produces proteolytic enzymes such as collagenase, phospholipase A, alkaline phosphatase, and acid phosphatase. Among them, collagenase is said to be a direct factor that decomposes collagen in periodontal tissue and leads to tissue destruction.

Therefore, in order to prevent and treat periodontal disease,
It is important to suppress the growth of Bacteroides gingivalis and further inhibit its pathogenic factors, and it is effective to apply substances with these functions in the oral cavity. From this viewpoint, the present inventors investigated a growth inhibitory factor of Bacteroides gingivalis and a collagenase inhibitory factor produced by this bacterium as an anti-periodontal disease agent.

It has been known that the growth of Bacteroides gingivalis is inhibited by antibiotics such as tetracycline and minocycline, and serum components, tetracyclines, metal salts, and the like have been reported as collagenase inhibitors. However, it cannot be said that it is appropriate to apply these substances in the oral cavity from the viewpoint of safety.

Therefore, when developing the product, we took into consideration the fact that it would be applied in the oral cavity, and that it is desirable to use a relatively safe natural product, and it is desirable to use a highly polar substance that has good affinity with saliva. As a result of intensive research, we discovered that tea catechins have a specific growth inhibitory effect on Bacteroides gingivalis and also have a collagenase inhibitory effect.
This has led to the completion of the present invention.

Tea catechins have been known to have physiological effects such as antioxidant activity, cholesterol increase suppressing effect, and blood pressure increase suppressing effect. However, the above-mentioned effectiveness of tea catechins is not known, and this was revealed for the first time by the present invention.

Since tea is drunk on a daily basis and its safety has been proven for a long time, it is thought that there is no need to worry about side effects due to continuous use. Therefore, by incorporating the product of the present invention into chewing cams, single-tablet candy, mouthwash, dental caries, toothpaste, and the like, it is possible to provide products that have a preventive effect on periodontal disease.

[Effects of the Invention] According to the present invention, tea is a natural product that has been used for a long time without any concerns about side effects, and specifically inhibits the growth of Bacteroides gingivalis, which is known as a periodontal disease pathogen. Furthermore, an anti-periodontal disease agent is provided which has the effect of inhibiting collagenase produced by this bacterium and is effective in preventing and treating periodontal disease.

[Example] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to the following Examples.

First, we will show a manufacturing example of the product of the present invention, then we will show a test example in which we tested the growth inhibitory effect of the product of the present invention on Bacteroides gingivalis and Streptococcus mutans, and then we will show the activity of the product of the present invention on the collagenase of Bacteroides gingivalis. A test example in which the inhibitory effect was tested is shown, and further examples of application of various foods using the product of the present invention are shown as examples.

Using 100g of crushed tea confectionery, make 80% ethanol solution 2.
000ml for 2 hours, and the extract was dried. The dried extract was diluted with water to a solid content of 10 to 30% and adsorbed on Toyopearl HW-40 gel beads for 1 to 2 hours. At this time, 1 for the volume 1j of gel beads
The amount of extract was 00 to 300 g. The gel beads that had adsorbed the extract were washed 2 to 3 times with 2 to 5 times the amount of water, and the gel beads were further washed 2 to 3 times with 2 to 5 times the amount of 15% ethanol aqueous solution. . The catechins adsorbed on the gel beads were released using an 80% ethanol aqueous solution, and the obtained tea catechins were freeze-dried to obtain the product of the present invention.

The product of the present invention (tea catechins) prepared by the method described above
The growth-inhibiting effect of Bacteroides gingivalis, a periodontal pathogen, and Streptococcus mutans, a caries-causing bacterium, was measured by the following method (antibacterial test).

Bacterial strain used: Bacteroides gingivalis FD
C381Bacteroides ginaivali
s ATCC33277Strep↑ococcus
1utans 6715 SMR3treptococ
cus IIutans Ingbritt test medium Trypticase soy agar Trypticase soy agar 40 g (Tri
DtiCaSe 5OyaQar') Hemin (hen
in) 5H menadione (ne
nedione) 0.5n.

Purified water 1ooo it plain heart infusion agar medium plain heart infusion agar
52 g (Brain hearing)
t1nfusion agar) purified water
1000 Il Trypticace soy broth 30 g (Tri
pticase say broth) hemin (hen
+in ) 5 ng menadione (
llenedione > 0.5rrrQ
Purified water 1000 ra Plain Heart Infusion Broth Medium Plain Heart Infusion Broth
37 g (Brain hea
rt 1nfusion broth) purified water
1000 l Test Method Bacteroides gingivalis was inoculated into Tryptigeis Soy Pross medium and Streptomyces mutans was inoculated into Plain Heart Infusion Pross medium and cultured at 37°C for 24 to 48 hours. O
D9. . The cells were boiled to a concentration of 1.0 and used as a bacterial solution.

Tryptigaceous soy aca medium (Bacteroides
Streptococcus mutans) or plain heart infusion agar medium (used for Streptococcus mutans) 101 and each bacterial solution 11 were mixed in a petri dish to prepare a test bacterial plate. A paper disc (ADVANTEC, Th
1ck 8nn) was placed on the test bacteria plate. 3
After culturing at 7°C for 24 hours, the presence or absence of an inhibition circle around the disk was confirmed, and the amount added to form the minimum inhibition circle was determined. Cultivation is carried out aerobically for Streptococcus mutans and anaerobically for Bacteroides gingivalis (
Co□:H,:N2=1:t:8).

The test results are shown in Table 1. In the table, Streptococcus mutans is abbreviated as S, nutans, and Bacteroides gingivalis is abbreviated as B, gingivalis. Tea catechins have a weak antibacterial effect against Streptococcus mutans, which is an oral cavity-causing bacterium, but have a strong antibacterial effect against Bacteroides gingivalis, a periodontal pathogen.

The inhibitory activity of Bacteroides gingivalis collagenase of the product of the present invention (tea catechins) was measured by the following method. To measure collagenase activity, use Collageno Kit C.
This was carried out using LN-100 (Cosmo Bio Co., Ltd.).

As a sample for inhibiting collagenase activity, the products of the present invention and tetracycline hydrochloride, which has been confirmed to have an inhibitory effect on collagenase activity, were used to compare their effects. For anaerobic culture, an anaerobic box is used and mixed gas (CO2: H2
:N2=1:1:8).

Test medium 1" J [1 added trypticase soy agar 40 g (Tri
pticase soy agar) hemin (hel)
lin) 5 B-menadione (n
enedione) 0.5ng horse defibrinated blood 100n+1 purified water
900n Trypticase Soy Broth 30g (Tri
pticase soy broth) hemin
lin) 5 11Q Menadione (
Menedione) 0.5 ng Purified water 1000 1 Collagenase preparation method The Bacteroides gingivalis collagenase solution used in this test was prepared by the following method.

Bacteroides gingivalis FDC381, which had been cultured anaerobically in a blood plate medium for 4 days, was inoculated into 1500 nl of a liquid medium for enzyme extraction, and cultured anaerobically at 37°C for 3 days. Centrifuge the culture solution (
The supernatant was saturated with 80% ammonium sulfate and centrifuged (12,000×G, 20 minutes) to collect an 80% ammonium sulfate fraction. 5 for both of these
0.05M Tris-HCl M containing nH Calcium Chloride
It was dissolved in buffer solution (pH 7.5) 3001, and thoroughly dialyzed using the same fresh solution. Filter the dialysis fluid (0.22μm
, HILLIPORE) L was used as a Bacteroides gingivalis collagenase solution.

Collagenase Inhibition Test Collageno Kit CLN-100 uses FITC-labeled collagen as a substrate and selectively denatures only the decomposed products generated after the reaction with collagenase at 35°C and then extracts with ethanol. Collagenase activity is quantified by measuring .

The experimental system includes Bacteroides gingivalis collagenase solution 200 μm and matrix collagen solution 200 μm.
, sample solution 100 μm, total 500 μm, 35°C
After a 2 hour reaction, the collagenase activity of each was measured (IU = 1 μg of collagen degradation/min).

The collagenase inhibitory activity of the sample was expressed as a percentage of the degree of decrease in collagenase activity upon addition of the sample, with the collagenase activity of the control set as 100. The test results are shown in Table 2. In addition, in the table, Bacteroides gingivalis is abbreviated as B and gingivalis.

The product of the present invention had a stronger inhibitory effect on Bacteroides gingivalis collagenase than tetracycline hydrochloride, which is known as a collagenase inhibitor.

Application Example Using the anti-dentistry agent (tea catechins) of the present invention prepared as described above, chewing gum, one-tablet candy, a mouthwash, and toothpaste were manufactured according to the following formulations.

Example 1: Chewing gum gum base sugar glucose starch syrup fragrance catechin Example 2 Candy sugar starch syrup citric acid tea catechins water Example 3 Confectionery sugar glucose Sucrose fatty acid ester tea catechins 2060% 55.0 15.0 9.3 0.5 0.2 100.0% 50.0% 34.0 1.0 0.2 14.8 100.0% 76.4% 19.0 0.2 0.2 Example 4: Cleaning agent ethanol fragrance copper chlorophyllin sodium saccharin Chlorhexidine hydrochloride tea catechins water Example 5: Training calcium carbonate glycerin carrageenan carboxymethylcellulose lauryl ethanolamide sucrose monolaurate copper chlorophyllin sodium Chlorhexidine hydrochloride saccharin 100.0% 30.0% 1.0 0.1 0.05 0.01 0.2 68.64 100.0% 50.0% 20.0 0.5 1.0 1.0 2.0 0.1 0.01 0.1 tea catechins water 0.2 25.09 100.0% 1st reward S of the product of the present invention (tea catechins) Antibacterial bacterial species against S. mutans and L. strain Minimum inhibition circle formation force (Pg/d:sk) mumu n5 67i55MR 1250 ngbritt 1250 L” Kamikaze 13 FDC381 ATCC332n +56

Claims (1)

[Claims] (1) Periodontal disease pathogen Bacteroides gingivalis (B
It is an anti-periodontal disease agent that has a specific growth inhibitory effect on the growth of Bacteroides gingivalis) and also has an inhibitory effect on collagenase produced by Bacteroides gingivalis, which contains gallic acid, (+)-catechin, (+)-gallocatechin, -)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate and dimers and trimers thereof, etc. in tea (Cameriasinensis)
An anti-periodontal disease agent characterized by containing extracted catechins as an active ingredient.